Background: Skin cutaneous melanoma (SKCM) is an aggressive malignant skin tumor. Ferroptosis is an iron-dependent cell death that may mobilize tumor-infiltrating immunity against cancer. The potential mechanism of long non-coding RNAs (lncRNAs) in ferroptosis in SKCM is not clear. In this study, the prognostic and treatment value of ferroptosis-related lncRNAs was explored in SKCM, and a prognostic model was established.Methods: We first explored the mutation state of ferroptosis-related genes in SKCM samples from The Cancer Genome Atlas database. Then, we utilized consensus clustering analysis to divide the samples into three clusters based on gene expression and evaluated their immune infiltration using gene-set enrichment analysis (GSEA) ESTIMATE and single-sample gene-set enrichment analysis (ssGSEA) algorithms. In addition, we applied univariate Cox analysis to screen prognostic lncRNAs and then validated their prognostic value by Kaplan–Meier (K-M) and transcripts per kilobase million (TPM) value analyses. Finally, we constructed an 18-ferroptosis-related lncRNA prognostic model by multivariate Cox analysis, and SKCM patients were allocated into different risk groups based on the median risk score. The prognostic value of the model was evaluated by K-M and time-dependent receiver operating characteristic (ROC) analyses. Additionally, the immunophenoscore (IPS) in different risk groups was detected.Results: The top three mutated ferroptosis genes were TP53, ACSL5, and TF. The SKCM patients in the cluster C had the highest ferroptosis-related gene expression with the richest immune infiltration. Based on the 18 prognosis-related lncRNAs, we constructed a prognostic model of SKCM patients. Patients at low risk had a better prognosis and higher IPS.Conclusion: Our findings revealed that ferroptosis-related lncRNAs were expected to become potential biomarkers and indicators of prognosis and immunotherapy treatment targets of SKCM. Skin cutaneous melanoma (SKCM), one of the most aggressive malignant skin tumors , is a coTraditional treatments have been the mainstream against advanced or metastatic melanoma in recent decades, although their aims are mainly to relieve symptoms and reduce tumor burden, with little help for prolonging survival. Due to the loss of early diagnosis and effective intervention, the identification of potential biomarkers for prognosis prediction and valid therapeutic targets of SKCM is urgently needed .As a breakthrough approach for metastatic melanoma, immunotherapy is based on the activation of the anticancer endogenous immune system, whose representative immune checkpoints are cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) . Howevervia multifarious signaling pathways, such as the inhibition of selenoprotein glutathione peroxidase 4 (GPX4)  . Recent 4 (GPX4) . NotablyNearly 98% of RNAs are non-protein coding RNAs (ncRNAs) . Long noIn this study, we established an 18-ferroptosis-related lncRNA prognostic signature of SKCM, which is expected to provide a reference for the follow-up diagnosis and treatment of SKCM.https://www.wikipathways.org). The study was free of the approval of the ethics committees for public access to the data acquisition. Subsequent data analyses were managed with the R (version 3.6.3) and R packages. A flowchart of the study is drawn in RNA-seq data containing 471 SKCM tumor samples were extracted, and somatic mutation data was downloaded from The Cancer Genome Atlas (TCGA) database. Forty ferroptosis-related genes were acquired from WikiPathways , an openp < 0.05 indicated statistical significance).After the preparation of the mutation annotation format (MAF) of mutation data, the mutation state of ferroptosis-related genes was evaluated in SKCM samples using the “maftools” R package, which supplies numerous analyses and visualization modules for cancer genomic studies . The mutvia the “pheatmap” R package.SKCM patients were unsupervised and classified into different subgroups based on the expression of ferroptosis-related genes using the “ConsensusClusterPlus” R package . The clip value <0.05.Gene set variation analysis (GSVA) enrichment analysis was performed to estimate the different Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between any two clusters using the “GSVA” R package, whose method is non-parametric and unsupervised to detect changes in pathway activation . The sigp < 0.05 indicated statistical significance. After finishing all the steps above, a single-sample gene-set enrichment analysis (ssGSEA) algorithm was utilized to quantify the components of different types of immune cells in the TME of SKCM.Moreover, the “ESTIMATE” R package  was usedp < 0.05 was used to filter differentially expressed lncRNAs (DElncRNAs), followed by intersection to obtain the common DElncRNAs, which are further displayed in the Venn diagram. Finally, univariate Cox analysis was applied to screen lncRNAs with prognostic value. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated. The results with p < 0.05 were selected for the subsequent analysis.Differential analyses between any two clusters were performed, as a standard of adjusted To test the predictive value of the prognostic lncRNAs obtained, unsupervised clustering was applied to the SKCM samples using the “ConsensusClusterPlus” R package once again. The clinicopathological characteristics in different subgroups are shown in heatmap plots, and Kaplan–Meier (K-M) curves were drawn to compare the survival outcomes between different clusters. In addition, the difference in transcripts per million (TPM) values of ferroptosis-related genes in each cluster was further explored.Multivariate Cox analysis was performed to screen out a prognostic signature constructing 18 prognostic lncRNAs in SKCM patients. According to the median of the risk score, the SKCM patients were separated into a high-risk group and a low-risk group.via the “Survival” R package to test the model’s clinical application value. The time-dependent receiver operating characteristic (ROC) curve assessed the predictive value of the model, and the area under the curve (AUC) was calculated using the “pROC” package. p value <0.05 indicated statistical significance. The 1-, 3-, 5-, 8-, and 10-year ROC curves of the model were painted. Additionally, the risk scores of SKCM patients in different survival states were also calculated to evaluate the model’s validity. The comparison between the two groups was performed via the Wilcoxon rank sum test, and p < 0.05 was considered statistically significant.The K-M survival curves of OS as well as stratification analyses for clinicopathological characteristics  in the high-/low-risk group were generated https://tcia.at/home) is an open access website containing the intratumoral immune landscapes and cancer antigenomes from 20 solid cancers. The immunophenoscore (IPS) is a scoring scheme for the quantification of tumor immunogenicity via machine learning. Considering the gene expression of four cell types  that determine immunogenicity, the IPS was calculated as z scores of the gene expression above, with higher scores associated with increased immunogenicity  database .To enhance the accuracy and persuasion of prognosis, the SKCM samples from TCGA database were randomly validated into training and validation sets twice to reverify the prognostic model. Once was divided from the middle, another time was at a ratio of 7:3. The chi-square test was used to verify the distribution difference between the training set and validation set, and https://www.wikipathways.org/index.php/WikiPathways; p < 0.05). The association between each ferroptosis-related gene was highly correlated, as shown in Melanoma is known as one of the most highly mutated malignancies . Having via the “ConsensusClusterPlus” R package. The clustering variable (k) was raised from two to nine, and k = 3 was chosen (p = 0.2148). Then, we performed GSVA enrichment analysis between different clusters to explore potential activated biological pathways , ImmuneScore (p = 5.2e−21), and ESTIMATEScore (p = 1.5e−19) were consistently as follows: cluster C > cluster B > cluster A. Additionally, the infiltration level of 23 kinds of immune cells in cluster C was the highest . The CDF and relative change in area under the CDF curve are shown in p < 0.0001). Additionally, the significant differences in TPM values of ferroptosis-related genes among the three geneclusters are displayed in p < 0.05). The significantly different results of the above analyses were all between the clusters, which indicated that the 77 lncRNAs possessed prognostic value.Based on the three clusters acquired from the previous steps, a differential analysis between any two of the clusters was conducted to screen out the significantly DElncRNAs as the standard of adjusted  diagram . FinallyUsing the 77 obtained prognostic lncRNAs, multivariate Cox analysis was utilized to construct a prognostic signature of SKCM composed of 18 prognostic lncRNAs . All SKCp < 0.0001). To evaluate the sensitivity and specificity of the prognostic signature, the ROC curves showed that the 1-, 3-, 5-, 8-, and 10-year AUCs were 0.73, 0.74, 0.76, 0.76, and 0.77, respectively (p = 4.6e−11). In addition, the difference in the mutation state of TP53 between the two groups was statistically significant (p = 0.0055), and the wild type was still the mainstream. To further verify the prognostic efficiency of the model, stratified survival analyses for clinicopathological information between the high- and low-risk groups were analyzed. The significantly different OS results of SKCM patients with different ages, sexes, tumor stages, T stages, N stages, M stages, TP53 expression, and wild-type TP53 levels between the high- and low-risk groups are shown in p < 0.05).The K-M curve revealed that the patients in the high-risk group had a poorer prognosis than those in the low-risk group (ectively . In the p < 0.05).Furthermore, the IPS data of SKCM was downloaded from the TCIA database. All four kinds of IPS showed significant differences between different risk groups, indicating that SKCM patients with low risk scores had better therapeutic effects .To verify the predictive ability of the prognostic model of SKCM patients, the SKCM patients were first randomly allocated from the TCGA dataset into a training set and validation set at 50% and 50% portion. The chi-square test was managed between the two sets, and there was no allocation difference , such as apoptosis and necroptosis, ferroptosis is recognized as a new form of PCD . Activatroptosis . The mutroptosis , while flncRNAs participate in the intricate regulation of ferroptosis and tumor immunity in various types of cancer cells , which hIn this study, we first analyzed the mutation state of ferroptosis-related genes in SKCM while TP53, ACSL5 and TF were the most frequently mutated genes. Missense mutation and C > T were the most common in SKCM. The C > T nucleotide transition has been reported to be mainly attributable to ultraviolet radiation . The mosThe tumor suppressor protein p53 (TP53) plays a critical role in the cellular response to various stresses. Depending on the levels of stress, TP53 could bidirectionally regulate ferroptosis in a context-dependent manner . SomaticACSL5, belonging to the acyl-CoA synthetase long-chain (ACSL) family, is a nuclear-coded pro-apoptotic gene that participates in cancer suppressors; however, it shows a pro-oncogenic role in gastric cancer . TF encoUsing ferroptosis-related gene expression, we divided SKCM patients into three clusters. Cluster C had the highest gene expression with the richest immune infiltration, which revealed that the expression of the clusters was significantly correlated with immune activation in the SKCM patients. Similarly, via univariate Cox regression analysis of the DElncRNAs selected from the clusters in SKCM patients. To test the prognostic abilities of the obtained lncRNAs, subsequent verified analyses were performed. Based on the expression of 77 prognostic lncRNAs, the SKCM patients were separated into three new clusters. Genecluster F had the most satisfactory overall survival rate, in which the ACSL family, SLC family, NCOA4, TF, and HMOX1 were upregulated, while GPX4 and TP53 were downregulated. In the review of ACSLs by + T cells downregulates the expression of SLC3A2 and SLC7A11 and results in a drag on cystine uptake, which finally leads to tumor cell ferroptosis. In genecluster F, SLC3A2 was significantly downregulated; however, SLC3A11 was specific.Furthermore, we identified 77 prognostic lncRNAs Then, we utilized multivariate Cox analysis to establish the 18-lncRNA prognostic model. Because of the risk score, all SKCM patients were grouped into the high- or low-risk group. The K-M curves of the overall and clinicopathological stratification survival outcomes between different risk groups were satisfied. The 10-year AUC of the prognostic model was 0.77. Furthermore, we successfully verified it in the validation set twice. Moreover, low-risk patients showed a better response to immunotherapy, which is consistent with previous immune results.After exploring published literature, the potential functions and clinical applications of AL606807.1, AC021078.1, AC004865.2, AC010245.2, AC018645.3, AC011511.5, AL021368.2, AC024909.1, AC100778.3, AC069222.1, AL592211.1, and KANSL1 L-AS1 have not been reported until now. Although our results indicated that ferroptosis-related lncRNAs might enhance immune activation, the role of lncRNAs in drug or immunotherapy resistance is seldom reported . SeveralThere are some limitations in our study. First, the training set and the validation set were taken from the same database. Unfortunately, we failed to collect external clinical data from independent cohorts. Second, all analyses in our study are descriptive, and further functional experiments are needed to explore the molecular mechanisms of these lncRNAs. Third, we mainly focused on the prognostic value of lncRNAs but lacked other relevant interactions. Therefore, a comprehensive overview of interactions between lncRNAs and related upstream and downstream factors is necessary.In this study, we conducted a novel prognostic 18-lncRNA signature for SKCM. Ferroptosis-related genes showed synergy with immune activation.


Objective: To investigate whether serum Tie-1 (sTie-1) is a valuable marker for predicting progression and prognosis of cervical cancer.Methods: Enzyme-linked immunosorbent assay (ELISA) was used to detect serum sTie-1 concentrations in 75 cervical cancer patients, 40 cervical intraepithelial neoplasia (CIN) patients, and 55 healthy controls without cervical lesions, and sTie-1 levels were compared between the groups. Receiver operating characteristic curves was used to evaluate the diagnostic value of sTie-1. The relationship between sTie-1 concentrations in patients with cervical cancer and clinicopathological features and prognosis were analyzed, and the risk factors for postoperative recurrence were determined using univariate and multivariable Cox proportional hazards regression.Results: We found that sTie-1 concentrations gradually increased according to lesion severity  and were significantly elevated in adenocarcinoma compared with healthy controls. sTie-1 levels strongly distinguished between cervical cancer patients and the healthy controls . Moreover, sTie-1 levels in cervical cancer patients were significantly associated with tumor size, advanced tumor stage, lymph node metastasis, and reduced 4-years progression-free survival. Cervical cancer patients with high sTie-1 concentrations had a 3.123-fold  higher risk for tumor recurrence.Conclusions: Elevated sTie-1 levels in patients with cervical carcinoma were associated with tumor progression and poor prognosis, indicating that sTie-1 may be a valuable marker for predicting progression and prognosis of cervical cancer. Cervical cancer is the fourth most common cancer in women and also is one of the leading causes of cancer related death in women . ApproxiRecently, the angiopoietin (Ang)/tyrosine kinase receptor (Tie) system, the second largest angiogenic system after vascular endothelial growth factor; (VEGF), has attracted increasing attention because targeting VEGF has demonstrated limited efficacy in cervical cancer detection and monitoring . Ang/TieMost studies on Tie-1 have focused on its function and related mechanisms within endothelial angiogenesis. In physiological angiogenesis, Tie-1 can bind directly to Tie-2 to form polymers, reduce the internalization of phosphorylated Tie-2 (pTie-2) and protein degradation , stabilize the phosphorylation signal for Tie-2, and positively regulate Ang/Tie-2 signaling to promote the survival and migration of endothelial cells . In tumoHowever, research on the expression of Tie-1 in tumors and its association with the clinicopathological characteristics and prognosis of tumor patients, as well as the mechanisms mediating its role in tumor progression, have not been well studied. A few studies have demonstrated that Tie-1 is highly expressed in tumor cells in some cases of invasive ductal carcinoma of the breast, and is positively associated with the rate of lymph node metastasis . In colop = 0.077). This study was approved by the hospital ethics committee at our medical center  and written informed consent was obtained from all enrolled participants. This research was conducted in accordance with the principles of the Declaration of Helsinki.We conducted a single-arm prospective study, with serum samples collected from 75 patients with cervical cancer, 40 cervical intraepithelial neoplasia (CIN) patients, and 55 healthy women (controls) presenting for primary care at the Department of Gynecology at the First Affiliated Hospital of Shihezi University School of Medicine between August 2017 and August 2020. The 75 cases of cervical carcinoma included 63 patients with cervical squamous cell carcinoma (CSCC) and 12 with cervical adenocarcinoma (CADC). The 40 cases of CIN included 17 cases of CIN2 and 23 cases of CIN3. Each diagnosis of cervical cancer was made by two or more clinicians  at the hospital. The prognostic value for sTie-1 in cervical cancer patients was evaluated according to the patients’ follow-up data as of October 2021. The median follow-up time was 30 months . Among the 75 patients, 18 had a recurrence and 10 died. Patients with inflammation , other types of cancer, connective tissue disease, or atherosclerosis  were excluded from the current study. The average age and age range of patients of normal group were 53  years, the average age and range of patients in the CIN group were 51  years, and the average age and range of cervical cancer group were 55  years. There was no significant difference in the age of the three groups by one way ANOVA , tumor size, pathological type, differentiation, whether there was lymph node metastasis (LNM), lympho-vascular space invasion (LVSI), and whether the infiltration depth of cervical stromal invasion exceeded 1/2. FIGO stage and tumor size were determined by two chief physicians of gynecological oncology, and the tumor size refers to the maximum diameter of the tumor. Among these patients, 46 patients with early stage (IA1-IIA2) underwent surgical treatment, and data on LNM, cervical muscular layer invasion, and LVSI were collected according to postoperative pathological examination report. For 29 patients with advanced cervical cancer, MRI results were used to determine whether LNM was present or whether cervical muscular layer invasion was greater than 50%. Patients were followed up according to the FIGO guideline. Progression free survival (PFS) refers to the time from the first day of treatment (surgery or radiotherapy or chemotherapy or concurrent chemoradiotherapy) to the date when there is evidence of tumor recurrence or the last follow-up. Overall Survival (OS) means the time that from the first day of treatment to death for any cause or to the date of last contact. The time is calculated by month. Patient characteristics were described in Serum samples from patients with CIN and cervical cancer were collected at first admission prior to any treatment. Briefly, fasting venous blood (3–4 ml) was collected in the morning for all participants. The samples were centrifuged at 3,000 G/min for 10 min. The sera were collected and the supernatants were extracted, placed in a 1.5 ml Eppendorf tube, and stored in a freezer at −80°C for future use. A standardized sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect sTie-1 concentrations with a human serum Tie-1 test kit purchased from RayBiotech . The assay was performed in strict accordance with manufacturer instructions; and each serum specimen was assayed twice. If the sTie1 values were discrepant by 10% or more, the samples were retested.p values < 0.05 were considered statistically significant.Statistical Package for the Social Sciences (SPSS) software  was used for all statistical analyses. Continuous variables not conforming to a normal distribution were described as medians and interquartile ranges. The differences in serum Tie-1 levels between the three aforementioned groups were analyzed using nonparametric tests, including the Kruskal–Wallis (K-W) test, the Jonckheere-Terpstra (J-T) test, and the Mann–Whitney (M-W) U test. Chi-square tests were used to compare rates between patients with cervical cancer and CIN. Receiver operating characteristic (ROC) curve analysis was performed to analyze the diagnostic value of sTie-1. Kaplan–Meier survival curves were used to predict patient survival. Cox univariate and multivariate proportional hazard models were used to determine the risk factors affecting the prognosis of patients with cervical cancer. All tests were two-tailed, and p < 0.05) (p < 0.001) (p = 0.011)  (p = 0.885) (p = 0.827) in the current study , 1,806.20 pg/ml , and 1,924.17 pg/ml , respectively. The K-W test showed that, sTie-1 levels gradually increased with an increase in the severity of cervical lesions ( < 0.05) . Moreove< 0.001) , and in = 0.011) , and in = 0.014) . However= 0.885) , and thent study .ROC curve analysis was performed to analyze whether sTie-1 could be used as a biomarker to effectively distinguish CSCC cases from CIN cases and healthy controls, as well as to distinguish CIN cases from healthy controls. We found that the area under the curve (AUC) values for sTie-1 [distinguishing CIN cases , cervicap values were all less than 0.05. Elevated Tie-1 concentration was no significant association with age, pathological type, differentiation, cervical stromal invasion and LVSI  (p = 0.215) (Patients were divided into high (n = 38) and low (n = 37) concentration groups according to the median sTie-1 level . In the group of patients with high sTie-1 levels, 13 patients had recurrence  and 7 died ; 4-years PFS and 4-years OS were 65.8 and 81.6%, respectively. In the low sTie-1 group, 5/37 (13.5%) patients experienced recurrence and 3/37 (8.1%) patients died; 4-years PFS and 4-years OS were 86.5 and 91.9%, respectively. PFS was shorter in the high sTie-1 group as compared with the low sTie-1 group  . Differe= 0.215) .p = 0.001), poor differentiation , and sTie-1 levels . We also observed a marginal association between PFS in patients with cervical cancer with a tumor size ≥2 cm . These factors were included in the Cox proportional hazards regression model in the multivariate analysis. The most important risk factors for tumor recurrence in cervical cancer patients were a FIGO stage ≥IIB, poor differentiated histopathology, and a high sTie-1 concentration, with adjusted hazard ratios of 5.161 , 2.996  and 3.123 , respectively  was 0.846, which was higher than that of sAng-2 (0.769) and the sAng-1:sAng-2 ratio (0.719), as reported in our previous study ; the corsistance . Thus, wIn addition to the substantial strengths of our study, we acknowledge some limitations. First, the sample size in the current study was insufficient, especially for examining associations with cervical adenocarcinoma; this may have led to some selection bias and reductions in statistical power. Second, the follow-up time for our patients with cervical cancer was relatively short. In the future, expanding the sample size of enrolled patients and healthy controls and extending the follow-up time could effectively validate our results and provide more conclusive, rigorous, and generalizable findings.Our findings revealed that, as the severity of cervical lesions increased, the level of serum Tie-1 gradually increased, and that sTie-1 levels can effectively distinguish cervical cancer patients from healthy controls. Thus, sTie-1 levels may be associated with the growth and progression of cervical cancer. We also found that high sTie-1 levels predict a poorer prognosis in cervical cancer patients. Therefore, sTie-1 may be a valuable biological marker for predicting the progression and prognosis of cervical cancer.


There are insufficient data in case of the potential association of habitual dietary L-arginine and the risk of type 2 diabetes mellitus (T2DM) incidence. Here we aimed to examine the potential effect of dietary L-arginine on the T2DM incidence.For this cohort study, 2139 T2DM-free adults from the participations of Tehran Lipid and Glucose Study (TLGS) were recruited. Follow up period was approximately 5.8 years. Daily intakes of protein and L-arginine were estimated using a validated food frequency questionnaire with 168 food item. Hazard Ratios (HRs) and 95% confidence intervals (CIs), adjusted for sex, age, smoking, diabetes risk score, physical activity levels, and total energy intakes as well as carbohydrate, fiber, fats and lysine, were calculated for L-arginine as both absolute intake and its ratio from total protein.Mean (±SD) age of the participants was 38.9 (±12.6) years and 54.6% were women. Mean (±SD) intake of dietary protein and L-arginine was 77.2 (±22.4) and 4.05 (±1.50) g/d, respectively. An increased risk of T2DM  was observed among participants with higher intakes of L-arginine (median intake of > 5.4 vs. 2.69 g/d). Total protein intake and the ratio of L-arginine to total protein intakes were not related to incidence of T2DM in both crude and adjusted models.We found that higher dietary L-arginine levels may increase risk of T2DM and it may have an independent role in T2DM development. L-arginine is a conditionally essential amino acid that involved in the synthesis of proteins, creatine, polyamines, agmatine, urea, and metabolism of proline and glutamate in the body , 2. The Short-term beneficial properties of L-arginine supplementation in some pathologic conditions including hypertension, hypertensive renal disease and cardiovascular disease have been investigated –9. SeverAlthough there are several animal studies investigated the effects of L-arginine supplementation on glucose and insulin homeostasis, long-term effects of L-arginine intake from usual diet in human is unclear. To the best of our knowledge, there is limited data in case of the association of habitual L-arginine intakes from diet and the risk of T2DM, therefore, in this study we aimed to evaluate the possible association of dietary L-arginine, as both absolute intake and its ratio from total protein intake, with the incidence of T2DM in a population-based study.This prospective cohort study was conducted using data collected from the Tehran Lipid and Glucose Study (TLGS). TLGS is an ongoing community-based cohort study, in a sample in the district 13 of Tehran, Iran, aimed to investigate and prevent non-communicable diseases . We recrn = 321), participants who had missing data of anthropometrics, biochemical values and physical activity (n = 63), and those with under- or over-report of total energy intakes  (n = 284), 2256 adults were remained and followed up to the fourth and fifth TLGS examinations, ~ 3 years apart. Mean period of follow-up was 5.8 years. Final analyses were conducted on 2139 adults , after exclusion of participants who had no follow-up after the baseline examination (n = 117). The flow chart of selection of study population is shown in Fig. Finally, after exclusion of subjects with T2DM diagnosis at baseline n = 32, particiThe study protocol was approved by the ethics research council of the Research Institute for Endocrine Sciences, Shahid Beheshti University of Medical Sciences. Moreover, written informed consents were obtained from all participants.2). Furthermore, waist circumference (WC) was measured to the nearest 0.1 cm, over light clothing, between the lower border of the ribs and the iliac crest at the widest portion. WC was measured using a soft measuring tape, with no pressure to the body. Waist to height ratio (WHtR) was calculated as WC divided by height (cm).Demographic data were conducted by the trained interviewers, using standard questionnaires. Measurements of demographic variables in TLGS have been reported elsewhere in details . Body weTo assess both systolic (SBP) and diastolic blood pressure (DBP) of participants, two measurements of blood pressure were taken, with at least a 30-s interval between two measurements. There was a 15-min rest before blood pressure measurements, and subjects were in the sitting position. For this purpose, we used a standardized mercury sphygmomanometer calibrated by the Iranian Institute of Standard and Industrial Research. Final blood pressure of participants was considered as the mean of the two measurements.To assess physical activity level of participants, the frequency and time spent on light, moderate, high and very high intensity activities, according to the list for common activities of daily life were asked over the past year. We used metabolic equivalent hours per week (METs h/week) to express physical activity levels.Participants had a 12–14 h of overnight fasting before biochemical measurements. Blood samples were drawn between 7:00 and 9:00 AM. Fasting serum glucose (FSG) and serum triglyceride (TG) were assayed using glucose oxidase and glycerol phosphate oxidase, respectively. The method used to measure FSG and TG was enzymatic colorimetric. High-density lipoprotein cholesterol (HDL-C) was measured after precipitation of the apolipoprotein B containing lipoproteins with phosphotungstic acid. Analyses were performed using Pars Azmoon kits  and a Selectra 2 auto-analyzer . Both inter- and intra-assay coefficients of variation of all assays were < 5%.Typical food intakes were assessed using a validated 168-item FFQ. The validity and reliability of the questionnaire have been previously evaluated . The intA T2DM patient considered as a subject who met at least one of the following criteria: (1) using anti-diabetic drugs, (2) fasting serum glucose (FSG) ≥126 mg/dL, (3) 2-h post challenging glucose (2-hPCG) ≥200 mg/dL .Mean and standard deviation (SD) values, and the frequency (%) of baseline characteristics were compared across tertiles of L-arginine intakes, using independent analysis of variance or chi square test, respectively. Hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between L-arginine intakes and L-arginine to protein ratio in relation to incidence of T2DM were estimated using Cox proportional hazards regression models with person-year as the underlying time metric. Cox models were adjusted for sex, age, smoking, diabetes risk score, physical activity levels, and total energy intakes as well as dietary carbohydrate, fiber, fats and lysine.The middle-time between the date of the first diagnosis of T2DM, and the most recent follow-up visit preceding the diagnosis were considered as the event date for T2DM cases. The difference between the calculated middle-time date and the date at which the subjects entered the study was considered as the follow-up time. In case of the censored and lost to follow-up subjects, the survival time was the interval between the first and the last observation dates. Follow-up duration and person-years were calculated using the measured survival time.P value< 0.05 being considered significant.All analyses were performed using IBM SPSS for Windows version 20, with a two-tailed 2 at baseline. A total of 143 cases of T2DM were identified over a median 5.8 years of follow-up. Mean (SD) intake of dietary protein and L-arginine was 77.2 (22.4) and 4.05 (1.50) g/d, respectively. There were no significant differences between dietary intakes of L-arginine, total protein and L-arginine/protein ratio in subjects with or without T2DM .Mean (SD) age of the participants was 38.9 (12.6) years and 45.4% were men. Mean (SD) BMI was 26.9 (4.7) kg/mP = 0.006), and had higher physical activity . There was no statistically significant difference in anthropometric and biochemical values as well as diabetes risk score across dietary intakes of L-arginine. Dietary intake of carbohydrate, fiber and protein was significantly increased with higher intakes of L-arginine, whereas intakes of total fat decreased .Baseline characteristics of participants are shown in Table .Association between L-arginine intakes as well as dietary protein and L-arginine to protein ratio with the incidence of T2DM after a 5.8 y of follow-up are shown in Table In the current prospective population-based study, we showed a potential adverse effect of high L-arginine intakes (more than 5.4 g/d) from habitual diet in relation to risk of T2DM, during 5.8 years of follow-up. Higher intakes of L-arginine however were not a potential risk for development of T2DM when it was considered in the context of total protein intake, as L-arginine-to-protein ratio.Mean dietary intake of L-arginine was 4.0 ± 1.5 g/d in our population, with major dietary sources of grains and meats. Although there is no recommended dietary allowance for L-arginine , 29. DifLimited data are available regarding the possible association of dietary L-arginine and the cardio-metabolic outcomes. Low intakes of L-arginine (below the median range of 3.8 g/d) has been reported to be associated with the higher C reactive protein (CRP) levels, while the highest level of L-arginine intake (> 7.5 g/d) increased risk of high-CRP levels by 30% . Also, hPatients with T2DM had higher plasma L-arginine concentration ; L-arginine concentration was positively associated with T2DM  . A 10-yeThe biological plausible mechanisms linking L-arginine intake to risk of T2DM are not well documented. One possible explanation may be related to induction of arginase activity by long-term high intakes of L-arginine , that haFurther prospective studies are needed to more fully determine the possible underlying mechanisms by which high intakes of L-arginine increased risk of T2DM.n = 143), we used the DRS in multivariate models, which allowed us to not adding many variables that would lead to instability of our models, and accounts for major T2DM confounders . However it had some limitations. First, we had not data on serum levels of L-arginine to consider in our analysis; however, an acceptable correlation was observed between dietary L-arginine intakes and serum L-arginine. Some inherent limitation points of observational studies including potential selection bias, information bias in measuring L-arginine intakes as study exposure, and non-differential misclassification of the exposure also should be considered. Furthermore, there are other potential confounding variables, including some dietary factors, which could affect the association of L-arginine and T2DM and were not included in our adjusted models; since L-arginine is derived from different food sources, other dietary factors occurring in the same foods could also have affected the findings. Other potential limitation of the present study is the possible changes of dietary patterns during the follow-up period, since we assessed the dietary information only at baseline examinations; however previous observations in our population indicates an acceptable stability of major dietary patterns over the time.The population-based prospective setting of the current study, and use of a validated FFQ to assess regular dietary intake are the main strength points of the study. Due to low number of outcomes (Our findings from this prospective study indicated that higher amount of dietary L-arginine may be potential risk factor for development of T2DM. Considering the increasing interest to ingestion of L-arginine as a popular dietary supplement, and also the limited data in case of the potential association between dietary L-arginine and cardio-metabolic outcomes especially T2DM, further cohort studies are required to clarify the possible association.


Pharmacological inhibition through XAV-939 as well as si-RNA mediated inhibition of β-catenin is initially effective in re-sensitization to therapy, however, prolonged inhibition shifts dependency from β-catenin to Notch signaling, with particularly high levels of receptors Notch 1 and Notch 2. The results are verifiable in a cohort of T-ALL patients comprising of responders vs. those who have progressed, with β-catenin, Notch 1 and Notch 2 elevated in progressed patients. Further, in patients-derived cells, silencing of Notch 1 or Notch 2 does not counter resistance to β-catenin inhibition, rather pharmacological pan-Notch inhibition is needed to overcome resistance and its effect on in vitro tumor sphere formations as well as in vivo liver metastases. Thus, wnt and Notch signaling are part of a regulatory loop mutually compensating for each other in T-ALL, while ensuring the maintenance of stem cell phenotype.Leukemia-initiating cells play critical role in relapse, resistance to therapies and metastases but the mechanism remains largely elusive. We report that β-catenin is over-expressed in almost all T-ALL patients and flow sorted β-catenin T-cell acute lymphoblastic leukemia (T-ALL) is aggressive, as its name suggests, progresses rather quickly. The T-cell variant of ALL accounts for about 15% ALLs in children and about 25% of ALLs in adults . AlthougThe goal of this study is to mechanistically dissect the role of β-catenin in stemness of T-ALL, particularly in patient-derived samples. We evaluated the role of β-catenin in inducing drug resistance and metastasis of T-ALL. Further, in view of the relapse observed in clinics, we mimicked such conditions to evaluate alternate signaling pathways that can compensate for the effective targeting of β-catenin. Our results indicate an intricate relationship between wnt and Notch signaling that helps maintain the stemness in T-ALL patients.The study involving human subjects was approved by the Institutional Review Board and the Ethics Committee at Sun Yat-sen University. A signed consent form was received from all patients and volunteers. The identifying information for T-ALL patients as well as healthy volunteers was not revealed to the researchers.Cells were treated with VDL  for 10 days. Control cells were left untreated. Cells were then harvested by centrifugation at 500 rcf for 5 min and re-suspended in DPBS  containing FBS at a final concentration of 2.5%. XAV-939 and BMS906024 used in the study were procured from Sigma (China).Active Beta Catenin Pathway Assay Kit  was used to quantitate β-catenin in patient-derived samples, following manufacturer’s suggested protocol. This qualitative ELISA kit is specifically designed to detect the active dephosphorylated form of β-Catenin. Notch family receptors were quantitated using specific ELISA kits for Notch1, Notch2, Notch3 and Notch4 . C-myc and Spi1 were also quantitated using specific ELISA kits purchased from Seajet Scientific, Shanghai, China.Single cell suspensions, made using GentleMACS kit , from patient samples were incubated in phosphate-buffered saline containing 1% FBS with human beta-Catenin APC-conjugated Antibody , and isotype-matched mouse immunoglobulins were used as controls. Samples were analyzed and sorted, using an EPICS ALTRA flow cytometer . For the positive and negative population, only the top 10% most brightly stained cells or the bottom 10% most dimly stained cells were selected, respectively.in vivo experiments, involving NSG mice , was approved by the Animal Research Ethics Committee at the Sun Yat-sen University. All methods were performed in accordance with the relevant guidelines and regulations. All mice were housed in sterilized animal facility. Based on our initial titrations, we injected 50,000 cells into the tail veins of NSG mice and euthanized mice after 7 weeks. The livers were visually inspected for signs of metastases.Our protocol for Samples from hepatic metastases were sectioned, conventionally stained with HE and immunohistochemically labeled with anti-cytokeratin using R-IHC and standard IHC and examined by a trained pathologist.Trypan blue dye exclusion assay was used to evaluate cell viability. Trypan blue  (0.4%) was added in 1:1 ratio with the single cell suspensions and the cells monitored and quantitated under bright field microscope. The live cells did not retain blue stain and their cytoplasm was clear while the dead cells were marked with blue cytoplasm.First, single cell suspensions were ensured enzymatically using trypsin and mechanically using a 22 gauge needle for 2 min. For the generation of tumorospheres, cells were plated in 24-well ultra-low attachment plates  at a density of 5,000 viable cells/well. Average sphere forming efficiency was evaluated after 18–21 days of culture under an inverted Olympus IX70 microscope camera  by counting spheres that were larger than 50 μm in diameter using the ImageJ Software.Trizol reagent  was used to isolate RNA following the manufacturer’s protocol. Quantitative RT-PCR reactions were performed on an ABI 7500 RT-PCR system (Applied Biosystems) using validated primers from BioRad (China). Only RNAse-free water was used throughout the assays.t-test and ANOVA were used to compare tested groups. Differences were considered significant when p < 0.05. Statistical analyses were conducted using Graphpad Prism software.All experiments were performed in triplicates. Two-tailed independent Student’s p < 0.0001) elevated in T-ALL patients, compared to controls , the sensitization was totally lost at 30 days . ClearlyTo ascertain whether increased β-catenin as well as Notch levels are relevant to T-ALL patients who have acquired resistance and whose disease has relapsed and/or progressed, we recruited T-ALL patients currently undergoing treatment at our Hospital. For this investigation, we identified 24 T-ALL patients (mostly diagnosed within the last 6 months) who were still responding to their assigned threrapies and another 28 patients whose disease had progressed and these patients had stopped responding to their last assigned therapy. We found β-catenin, Notch1, Notch2 as well as c-Myc to be significantly over-expressed in patients whose disease had progressed .in vivo in a mice metastasis model. Cells from control condition, si-β-catenin and si-β-catenin + BMS906024 were injected in tail veins of mice and as seen in Next, we identified 2 patients with progressed disease and the highest expression of β-catenin. Tumor cells were isolated, single cell suspensions made and the cells cultured in the lab. We now wished to study the inter relationship between wnt and Notch signaling, with regards to the maintenance of stemness. In assays performed on cells from one patient, VDL resistance was observed as expected (because of increased β-catenin) . FurtherEven though wnt signaling is often de-regulated in hematological malignancies , its rolNotch1 mutations (The relevance of Notch signaling in T-ALL is well regarded . A role utations . Not jusutations .In the report on role of stabilized β-catenin in development of T-ALL, up-regulation of c-Myc was reported as a consistent secondary event . MoreoveBased on our observations, we propose that future investigations should evaluate simultaneous inhibition of multiple signaling pathways, particularly those that are closely related. Even though we investigated Notch signaling as an alternate pathway when β-catenin was inhibited because of its relevance to T-ALL and the probability of taking up almost all the functions, particularly those connected to stemness, our unpublished preliminary results from high throughput assays indicate that Notch family members indeed are among the top potential candidates that can control the proliferation and metastasis of T-ALL cells when β-catenin inhibition is rendered ineffective.A number of drugs that target either Notch signaling or β-catenin are progressing through the clinical trials . This isThe original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Institutional Review Board and the Ethics Committee at Sun Yat-sen University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Animal Research Ethics Committee at the Sun Yat-sen University.LZ, JW, YF, BK, ZF, and J-JL participated in performing experiments, collecting data, and analyzing results. JG, BT, RX, and GP analyzed data and performed statistical analyses. J-JL conceptualized and supervised study. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous research demonstrated a relationship between ON and disordered eating symptoms  and disordered eating attitudes (DEA). Since screening for orthorexia nervosa is now part of clinical practice, the measurement instruments to be used must be clinically significant, reliable, valid and sensitive to our target population. The main objective of the present study was to confirm the factor structure of the Arabic version of the ORTO-R using a first sample of Lebanese adults and confirm those results on another sample. The secondary objective was to assess sex differences in terms of ON and DEA, as well as to examine whether symptoms of ON were related to DEA in Lebanese adults.A total of 783 Lebanese adults was selected to participate in this cross-sectional study (January-May 2018) using a proportionate random sample from all Lebanese governorates.2. All items of the ORTO-R were extracted during the factor analysis and yielded a two-factor solution with Eigenvalues > 1 . This factor structure was confirmed by a confirmatory factor analysis; the Maximum Likelihood Chi-Square  =  26.894 and Degrees of Freedom  =  8, which gave a χ2/df  =  3.36. The Tucker Lewis Index (TLI) value was 0.914, whereas the standardized root mean square residual (SRMR) value was 0.032. The root mean square error of approximation (RMSEA) value was 0.077 [95% CI 0.046–0.111] (pclose = 0.07) and comparative fit index (CFI) value was 0.967 respectively, indicating a good fit of the model. There was also no measurement invariance between genders. Female gender was significantly associated with lower ORTO-R scores (more orthorexia nervosa) compared to males . However, no significant difference was found between genders in terms of EAT-26 scores . Higher ORTO-R scores  were significantly related to higher EAT-26 total scores (higher levels of DEA) as well as higher dieting, bulimia and oral control scores in both females (from a weak to a moderate positive correlation) and males (a weak positive correlation).The mean age of the total sample was 27.78 ± 11.60 years (Min. 18 –Max. 84)  and their mean BMI was 24.36 ± 5.31 kg/mThis cross-sectional population-based study confirmed the factor structure of the Arabic version of the ORTO-R, demonstrated an association between ON and DEA and revealed more ON among females compared to males. Not yet classified in the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5)  or in thPrevious research demonstrated a relationship between ON and disordered eating symptoms  and disordered eating attitudes (DEA) –13. DEA Several socio-demographic factors have been correlated with ON , 20. A sScientific interest in ON has resulted in an increased amount of ON measures. Nowadays, there are ten distinct ON measures , includiSince screening for orthorexia nervosa is now part of clinical practice, the measurement instruments to be used must be clinically significant, reliable, valid and sensitive to our target population . Nowadayth World Medical Association General Assembly, Helsinki, Finland) and its later amendments or comparable ethical standards. Written informed consent was obtained from all participants. Participants received no financial incentive.The present study has been approved by the ethics committee at the Psychiatric Hospital of the Cross. All procedures performed in our study were in accordance with the 1964 Helsinki declaration   using a proportionate random sample from all Lebanese governorates. The latter is divided in sub-districts, which are also divided in villages. In each selected village, the questionnaire was distributed randomly to the households, based on random sampling technique to select the included house. All members in the household, if eligible, were invited to participate in the study; those who accepted our invitation were asked to fill out the questionnaire. Persons suffering from a clinical mental impairment (as reported by a family member) affecting their ability to understand the questions were excluded from the study. The same methodology is described elsewhere in papers from the same project , 29–32.  EAT-26) .A total of 60 participants was deemed necessary the factor analysis of the ORTO-R scale  , whereas2, normal weight between 18.5 and 24.9 kg/m2, overweight between 25 and 29.9 kg/m2, and obesity ≥ 30 kg/m2.Demographic information was gathered, including participants’ self-reported sociodemographic characteristics such as age, sex and anthropometry . BMI was categorized according to the World Health Organization (WHO) recommendations: underweight <18.5 kg/mThe ORTO-R , the revThe EAT-26  was desiSPSS version 25 was used to perform the statistical analysis. The total sample was randomly divided into two subsamples, using the SPSS option; the first sample, used to conduct the factor analysis, consisted of 388 participants  females), whereas the second sample, used to conduct the confirmatory factor analysis, consisted of 395 participants  females). To confirm the ORTO-R construct validity, a factor analysis (FA) using the principal analysis component was run on sample 1. To ensure the model’s adequacy, the Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy and Bartlett’s test of sphericity were calculated. Factors with an Eigen value >1 were retained. Moreover, Cronbach’s alpha values were recorded for reliability analysis for the total scale and its subscales. Second, a confirmatory factor analysis (CFA) was carried out on Sample 2 using SPSS AMOS v.24. The root mean square error of approximation (RMSEA) statistic, standardized root mean square residual (SRMR), the Tucker Lewis Index (TLI) and the comparative fit index (CFI) were used to evaluate the goodness-of-fit of the model as these are the most commonly used indices .The normality of distribution of the ORTO-R and EAT-26 scores was confirmed via a calculation of the skewness and kurtosis; values for asymmetry and kurtosis between -1 and +1 are considered acceptable in order to prove normal univariate distribution . These cp<0.001; αCronbach = 0.755)  .A first confirmatory factor analysis was run, using the one-factor structure obtained in the original version . The folA second confirmatory factor analysis was run on sample 2, using the structure obtained in sample 1 (two-factor solution). The following results were obtained: the Maximum Likelihood Chi-Square  =  26.894 and Degrees of Freedom  =  8, which gave a χ2/df  =  3.36. The TLI value was 0.914, whereas the standardized RMR value was 0.032. The RMSEA value was 0.077 [95% CI 0.046–0.111] (pclose = 0.07) and CFI value was 0.967 respectively, indicating a good fit of the model. The standardized factor loadings of the two-factor model of the Arabic version of the ORTO-R model are presented in 2 = 39.5 and df = 20, whereas for the fully constrained model, we obtained the following values: χ2 = 39.6 and df = 22 (p = 0.951)  (pclose = 0.741), showing that we have configural invariance. We also obtained metric invariance between genders as follows: for the unconstrained model, we obtained the following values: χ= 0.951) . Accordi= 0.951) , we have2 = 0.006). However, no significant difference was found between genders in terms of EAT-26 scores .Female gender was significantly associated with lower ORTO-R scores compared to males  nor with the total EAT-26 score . Higher BMI was significantly but weakly associated with ORTO-R  and EAT-26  scores.Higher ORTO-R score  was significantly related to higher EAT-26 total score (higher level of DEA) as well as higher dieting, bulimia and oral control scores in both females (from a weak to a moderate positive correlation) and males (a weak positive correlation) .Cronbach = 0.755). The ORTO-R was also moderately correlated to the EAT-26 total score, confirming the convergent validity of the scale. This demonstrates that the Arabic version of the ORTO-R can be considered a reliable ON screening tool for the Lebanese population. To the best of our knowledge, only one study [The present study aimed at investigating the psychometric properties of the Arabic version of the ORTO-R scale in a general population sample. The factor analysis yielded a two-factor solution in the Arabic version of the ORTO-R, which later confirmed by a CFA with a satisfactory goodness-of-fit. Reliability analysis of the ORTO-R across the whole questionnaire showed adequate internal reliability . Our results demonstrated that females were more likely to have ON thoughts and behaviors than males. Recent Lebanese studies , 47, 48 The Lebanese culture is characterized by a collectivist culture . StudiesThe present results also indicated that no significant difference was found between women and men in terms of DEA, in opposite to previous studies , 58 thatThe present study has some limitations. Firstly, the cross-sectional design does not allow the assessment of test-retest reliability of the Arabic version of the ORTO-R. Secondly, a cross-sectional design does not allow us to infer causality even if our findings demonstrated association between ON and DEA. The ORTO-R was measured in the current study using a four-point Likert scale . A seleThis cross-sectional population-based study confirmed the factor structure of the Arabic version of the ORTO-R, demonstrated an association between ON and DEA and revealed more ON among females compared to males. The Arabic version of the ORTO-R seems to be an adequate instrument for ON screening among Lebanese adults, with more research needed to confirm our results. The link between ON and DEA is not yet completely understood. Future longitudinal studies are warranted for this purpose.


DAC exerted no impact on hematopoietic reconstitution. However, patients who were treated with the high-dose DAC schedule had significantly higher incidence of overall survival  and leukemia-free survival , and lower incidence of relapse (41.1%) and grade II–IV acute graft versus host disease  at 3 years, when compared with those treated with standard conditioning regimens or with the low-dose DAC schedule. In conclusion, high-dose DAC combined with standard conditioning regimens before allo-HSCT is feasible and efficient and might improve outcomes of patients with relapsed or refractory AML, which provides a potential approach to treat these patients.Hypomethylating agents, decitabine (DAC) and azacitidine, can act as prophylactic and pre-emptive approaches after allogeneic hematopoietic stem cell transplantation  and a non-intensive bridging approach before allo-HSCT. However, they are rarely used as a part of conditioning regimens in patients with relapsed or refractory acute myeloid leukemia (AML). This retrospectively study included a total of 65 patients  with relapsed or refractory AML who were treated by allo-HSCT after myeloablative conditioning regimens without or with DAC (high-dose DAC schedule, 75 mg/m With significant advances in the treatment of acute myeloid leukemia (AML), 60%–80% of AML patients can achieve complete remission (CR) after standard induction chemotherapy consisting of anthracyclines and cytarabine . HoweverTheoretically, minimizing the disease load before allo-HSCT is the most effective way to decrease the relapse rate. Hypomethylating agents (HMA), decitabine (DAC) and azacitidine, are DNA methyltransferase inhibitors that can inhibit DNA methyltransferase, reverse DNA methylation, and reactivate tumor-suppressor genes in leukemia cells . MeanwhiPrevious studies have investigated the feasibility and effectiveness of using HMA as a bridging therapy prior to transplantation in patients with myelodysplastic syndrome (MDS) and/or AML . In thisSixty-five patients with relapsed or refractory AML from April 2006 to May 2016 were included in this retrospective study. All patients were diagnosed according to the 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia . PatientThis study was approved by the ethical committee of the 5th Medical Center of Chinese PLA General Hospital (No. KY-2019-12-57). Informed consents were obtained from all of the patients or from a parent or guardian of the patients under 16 years old, in accordance with the Declaration of Helsinki.2 on day −9 and 50 mg/m2 on day −8 intravenously for 3 h). Patients in the LDD-SC group received the standard preparative regimen (as described above) plus low-dose DAC (25 mg/m2/day on day −10 to −8 intravenously for 3 h).All patients received the myeloablative conditioning regimens. Patients in the SC group received BU plus CY or CY plus TBI. BU/CY was administered as follows: BU 0.8 mg/kg q6h intravenously on day −7 to −4, CY 60 mg/kg/day on day −3 to −2. CY/TBI was administered as follows: CY 60 mg/kg/day on day −4 to −3, TBI 5.0 Gy/day on day −2 to −1. Two different DAC doses were determined based on previous studies –15. Pati2 on day +1, 10 mg/m2 on day +3 and day +6). Patients receiving HLA-mismatched sibling grafts were given antithymocyte globulin  combined with CSA and a short course of MTX. Patients receiving unrelated grafts were given a regimen consisting of CSA, a short course of MTX, MMF (1.5 g/m2/day on days 0–28), and CD25 antibody .Patients receiving HLA-matched sibling grafts were given cyclosporin A  combined with a short course of methotrexate  was prevented by a combination of alprostadil, ursofalk, and heparin.All patients were cared for in sterile laminar flow wards. Ganciclovir and trimethoprim sulfamethoxazole were routinely administered to prevent viruses and pneumocysis carinii infection, respectively. Patients without invasive fungal infections (IFIs) were administered with fluconazole to prevent fungal infections, while those with IFI were administered with primary effective antifungal drugs. Patients with platelet counts <20 × 10-free survival (LFS) was defined as the time from transplantation to the date of relapse or death of any cause. Overall survival (OS) was defined as the time from transplantation to death of any cause or last follow-up. Relapse was defined as BM blasts ≥5%, appearance of leukemia cells in peripheral blood, or extramedullary leukemia cell infiltration after CR. Acute GVHD (aGVHD) was diagnosed and graded according to the criteria of Przepiorka et al. , and medians were used to describe data with non-normal distribution. Multivariate analysis was performed to calculate hazard ratio (HR) and 95% confidence interval (CI) by using Cox proportional hazard regression models. For all analysis, p < 0.05 in two-tailed test was considered as statistically significant. All statistical analyses were performed with SPSS 20.0.The median ages of patients in the SC group, the HDD-SC group, and the LDD-SC group were 36 , 42 , and 35 , respectively. The median follow-up times after transplantation were 9 months , 40 months , and 5 months , respectively. Patient characteristics are summarized in The median times to neutrophil engraftment in the SC group, the HDD-SC group, and the LDD-SC group were 16 days , 15 days , and 16 days  (p < 0.05).CR at 3 months after allo-HSCT was 75.0%, 57.1%, and 63.0% in the HDD-SC group, the LDD-SC group, and the SC group, respectively. The cumulative incidence of OS at 3 years in the HDD-SC group (50.0%) was significantly higher than that in the LDD-SC group (22.2%) or the SC group (18.5%) (p < 0.05), while there was no significant difference between the LDD-SC group and the SC group (p > 0.05)  of patient in the SC group died of leukemia relapse (n = 21) and sudden cardiac death (n = 1); 50.0% (10/20) of patients in the HDD-SC group died of severe infections , leukemia relapse (n = 4), respiratory failure (n = 1), and sudden cardiac death (n = 1); and 77.8% (14/18) of patients in the LDD-SC group died of leukemia relapse (n = 9), severe infections , and severe aGVHD (n = 1).p < 0.05) (Sixty-three percent (17/27) of patients in the SC group developed aGVHD, 14 of whom were grades II–IV and 6 of whom were grades III–IV. Forty percent (8/20) of patients in the HDD-SC group developed aGVHD, two of whom were grades II–IV and none were grades III–IV. Of the patients in the LDD-SC group, 55.6% (10/18) developed aGVHD, seven of whom were grades II–IV and two of whom were grades III–IV. The incidence of grades II–IV aGVHD in the HDD-SC group (10.0%) was significantly lower than that in the LDD-SC group (45.3%) or the SC group (53.5%) , 5.0% (1/20), and 44.4% (8/18) of patients were observed in the SC group, the HDD-SC group, and the LDD-SC group, respectively.In a multivariate analysis of patients in the SC group and the HDD-SC group  and a low-dose schedule (25 mg/m2/day × 3) of DAC, as part of standard conditioning regimens, were compared in allo-HSCT patients with relapsed or refractory AML in this study. We found that the two dose schedules had no impact on hematopoietic reconstitution when compared with the standard conditioning regimens (BU/CY and CY/TBI). In addition, the high-dose DAC schedule, but not the low-dose DAC schedule, provided significantly longer OS and LFS and significantly lower relapse and grade II–IV aGVHD. Multivariate analyses also suggested that high-dose DAC was associated with longer OS, lower relapse, and lower grade II–IV aGVHD. Compared to the FLAMSA-RIC regimens  in HSCT patients with relapsed or refractory AML or MDS, the high-dose DAC schedule in this study showed a higher 3-year OS and 3-year LFS , although the 3-year OS and 3-year LFS in the SC group were lower   , which s. 23.7%) , 30, esp. 23.7%) .Relapse remains as a main cause of treatment failure. The longer OS in the HDD-SC group in this study might be due to the longer median time to relapse (11 months), which is consistent with a previous report that patients who relapse <6 months after allo-HSCT have higher risk of mortality . Thus, eAs for causes of death in this study, infection was the leading one in the HDD-SC group and the LDD-SC group , which was consistent with a previous study  conducted in the similar cohort using DAC plus myeloablative allo-HSCT . The higThere are limitations in this study. First, the results presented here should be interpreted with caution due to the retrospective characteristic of the study and the limited number of patients in each group. Second, the imbalance and relatively short follow-up time of some patients might bias the results. Therefore, a prospective study, which includes multiple centers and more relapsed or refractory AML patients, will be conducted for confirmation of the promising observations. Dose schedules of DAC, as part of standard conditioning regimens before allo-HSCT, will be optimized to make a better balance between its side effects and antileukemia activities to further improve treatment outcomes. Nevertheless, our results show at least the feasibility and efficacy of DAC-involved standard conditioning regimens before allo-HSCT in patients with relapsed or refractory AML.In summary, high-dose DAC combined with standard conditioning regimens before allo-HSCT is feasible and efficient and might improve outcomes  in patients with relapsed or refractory AML, which is suggested to be a potential approach to treat these patients.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by the ethical committee of the 5th medical center of Chinese PLA general hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin. Written informed consent was obtained from the individual(s), and minor(s)’ legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.YL and LC designed and performed the study and collected and analyzed the data. CX, JC, JH, NL, SL, JX, TS, LW, YZ, and YS collected and analyzed the data. SC and LH designed the study and drafted and revised the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the Capital Characteristic Clinical Project of China (Z171100001017188).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Subsequently, we performed a replication study in an independent Chinese cohort for selected variant. A meta-analysis combined the previous GWASs with the replication study was also conducted. The epigenetic annotation and cytokine assay were used for exploring potential variant function.We systemically extracted the genetic data of p = 8.69E-03). The estimated effect size for this SNP was larger in Asian population than that in European population . The cytokine assay also showed that the risk allele T of the SNP rs2431697 is inversely associated with plasma TNF-α levels in health controls (p = .016).The extracted genetic association data from three previous GWASs showed that the allele T of functional SNP rs2431697 increased RA susceptibility. The significant association for the SNP was also found in the Chinese replication cohort (OR = 1.24, 95% CI = 1.06–1.46, miR-146a gene is associated with RA risk.miR-146a gene and susceptibility of RA was unclear.The association between SNPs in We investigated the genetic association using GWASs data and a replication study.miR-146a gene is associated with RA risk.The SNP rs2431697 in In summary, this study supports that genetic variant in  AlthougmiR-146a gene and susceptibilities of several autoimmune diseases. Previous studies have suggested that the risk allele of rs2910164 is associated with increased risk of ankylosing spondylitis [miR-146a and risk of systemic lupus erythematosus (SLE) in Asian population. The risk-associated G allele had decreased binding to transcription factor Ets-1, contributing to reduced levels of miR-146a in SLE patients. Then, the genetic association of rs2431697  with SLE was replicated in a case-control study in European population. Gene expression analysis revealed that the SNP rs2431697 is not associated with PTTG1 expression levels, but with the miRNA-146a, where the risk allele correlates with lower expression levels of the miRNA [miR-146a-target gene regulation within the type I interferon pathway.MicroRNA-146a (miR-146a), a small non-coding RNA, is considered to be an important negative feedback regulator of innate immune responses and autoimmunity . Recentlndylitis . Jazdzewndylitis  identifihe miRNA . Similarhe miRNA  found thmiR-146a gene polymorphisms with RA susceptibility. However, the review showed that only the SNP rs2910164 has been previously reported for RA susceptibility. Furthermore, no significant association for the SNP in the meta-analyses (p > .05) was found [miR-146a are increased in peripheral blood mononuclear cells [miR-146a gene and RA risk using the data of previous genome-wide association studies (GWASs) and a new replication study.Recently, systematic review and meta-analyses investigated the association of as found ,13. In car cells , CD4+ T ar cells  and RA sar cells  in RA paar cells  and decrar cells . Given t2.2.1.http://jenger.riken.jp/en/result). All the subjects in GWAS_CN were recruited from Anhui Province of China [The available data from three previous GWASs  were used ,22. The An independent replication cohort in Anhui (REP_AH: 779 cases and 1809 controls) was further recruited for selected SNP. During the study period (January 2014–June 2019), all questionnaires were obtained from the First Affiliated Hospital of Anhui Medical University or Anhui Provincial Hospital. The included RA patients were determined according to the revised criteria of the American College of Rheumatology 1987 criteria . All pat2.2.The details of genome-wide quality control and analyses for the three GWASs were available in previous studies ,22. For 2.3.p < .05) in controls will be conducted for quality control. We performed association analysis for REP_AH based on an additive model using PLINK version 1.07 (http://zzz.bwh.harvard.edu/plink/) [Selected SNP was genotyped in the independent cohort (REP_AH) using SNPscan technology . Hardy–Weinberg equilibrium (HWE) test (/plink/) . The sta/plink/) .2.4.Q and I2 statistic tests simultaneously.The three GWASs  and the replication cohort (REP_AH) were then meta-analysed with the inverse-variance method under a fixed-effect model using Stata version 14.2 . We conducted meta-analyses in Asian and trans-ethnic population, respectively. We described study heterogeneity using the 2.5.r2 > 0.8), as implemented in HaploReg version 4.1 [To further estimate potential regulatory roles of the likely causal variants in associated loci, we annotated lead SNP along with its surrounding correlated SNPs . The association results of selected SNPs (rs57095329 and rs2910164) in each GWAS are also listed in We firstly investigated genetic association of ach GWAS . The T a3.2.Supplementary Table S1. As expected, significant association was found in REP_AH . The p value for HWE test in controls was .082. Moreover, we performed a subgroup analysis of the relationship between SNP rs2431697 and RA in serologically positive patients in the replication cohort . The effect size between rs2431697 and RA risk was still stable . In this replication study , the statistical power will be 72.5%. Next, we performed an expanded meta-analysis with all results in the three GWASs and replication association study (REP_AH). The data synthesized in meta-analyses are presented in p = 4.37E-07) in Asian population and 1.07  in trans-ethnic population. The effect size of the SNP rs2431697 was larger in Asian than that in European. No significant heterogeneity was observed across Asian population . In contrast, a significant heterogeneity was found in the trans-ethnic meta-analysis .To further validate the identified association, we selected the SNP rs2431697 in the replication study. The clinical and demographic features of the included subjects in the replication study are shown in 3.3.r2 > 0.8), as implemented in HaploReg version 4.1. The data showed that the SNP rs2431697 was overlapped with multiple epigenetic marks in immune cells (Supplementary Table S2). It should be noted that the SNP is mainly overlapped with the enhancer marks in these cells. In addition, the cytokine assay also showed that the T risk allele of rs2431697 is inversely associated with plasma TNF-α levels in health controls (p = .016)  .4.miR-146a gene and susceptibility of RA. We firstly identified that the T allele of the functional SNP rs2431697 increases RA risk. The effect direction of the SNP was consistent in the discovery and replication association study  but not in Asian meta-analysis .In this study, we systemically estimated the association between SNPs in on study . FurthermiR-146a [miR-146a expression in CD4+ T cells is positively correlated with the levels of TNF-α in both SF and peripheral blood of RA patients, but not associated with the levels of other cytokines. We further speculated that the inverse association might be attributable to reverse causality since in vitro studies showed that TNF-α stimulation  upregulated miR-146a expression in Jurkat T cells and human CD4+ T cells in a dose-dependent fashion [Of note, the cytokine assay in this study also showed that the T risk allele of rs2431697 is inversely associated with plasma TNF-α levels in healthy controls. We judged that the inverse correlation may not derive from common confounding bias since the possible factors  were included as covariates in the regression model. Although previously functional study showed that miR-146a overexpression can significantly reduce TNF-α levels in the supernatant of the cell culture medium of THP-1 cells following LPS treatment [miR-146a . Consist fashion ,32.miR-146a gene and susceptibility of RA in different populations. Second, the initial genetic findings were replicated in an independent genotyped data, which strengthened the validation of the conclusion. Meanwhile, there are, however, also several limitations. First, the unavailable individual data in previous GWAS studies may limit conditional association analyses in meta-analysis level [The following advantages of this study ensured the credibility of the results. First, by using large-scale GWASs data, we can systemically estimate the association between SNPs in is level ,22. SecomiR-146a gene correlates with susceptibility of RA. The detailed mechanism of the loci in the pathogenesis of RA needs to be further studied.In summary, our data firstly supported that the SNP rs2431697 in Click here for additional data file.


The nanostructure-based surface texturing can be used to improve the materials wettability. Regarding oil–water separation, designing a surface with special wettability is as an important approach to improve the separation efficiency. Herein, a ZnO nanostructure was prepared by a two-step process for sol–gel process and crystal growth from the liquid phase to achieve both a superhydrophobicity in oil and a superoleophobic property in water. It is found that the filter material with nanostructures presented an excellent wettability. ZnO-coated stainless-steel metal fiber felt had a static underwater oil contact angle of 151.4° ± 0.8° and an underoil water contact angle of 152.7° ± 0.6°. Furthermore, to achieve water/oil separation, the emulsified impurities in both water-in-oil and oil-in-water emulsion were effectively intercepted. Our filter materials with a small pore (~5 μm diameter) could separate diverse water-in-oil and oil-in-water emulsions with a high efficiency (>98%). Finally, the efficacy of filtering quantity on separation performance was also investigated. Our preliminary results showed that the filtration flux decreased with the collection of emulsified impurities. However, the filtration flux could restore after cleaning and drying, suggesting the recyclable nature of our method. Our nanostructured filter material is a promising candidate for both water-in-oil and oil-in-water separation in industry. Surface wettability is an important material property, and it is determined by surface structure and surface chemical composition ,2. BasedFurthermore, the structural parameters of filter membranes may play pivotal roles in the performance of oil–water separation, such as pore size, porosity, and membrane thickness ,22,23. TAmong these structural parameters, the diameter of the liquid distributor and the hole shape dominated the oil–water separation. To achieve a high demulsification efficiency, the filtering precision should be improved accordingly ,29. For In addition, membrane filtration modified by organic polymer could achieve effective separation of oil–water mixtures. Due to its poor stability in the aqueous medium, however, it is hard to use the common polymer membranes with a special wettability in practice . TherefoZnO nanostructured pillars could be used in complex industrial oil–water mixtures ,34,35,362Zn (0.75 M (CH3COO)2Zn)  and Monoethanolamine ((CH3COO)2Zn and Monoethanolamine in the proportion 1:1)  were dissolved in a 2-methoxyethanol solution . This solution was magnetically stirred and heated in a temperature-controlled water bath . The seed solutions were obtained by standing at room temperature for 24 h. Substrates are submerged in the seed solution and lifted repeatedly. Finally, the substrates with the ZnO nanostructured coating were sintered  to stabilize the coating.The stainless-steel fiber felts with the filtration precision of 5 µm were used as a substrate in this study. To eliminate surface contamination, all substrate samples were ultrasonically cleaned with absolute ethanol and ultra-pure water for 10 min. (CH3COO)3)2 /hexamethylenetetramine (HMTA) /DAP  (0.05 M Zn(NO3)2, Zn(NO3)2, and DAP in the proportion 1:6) aqueous solution (10 min) and then was heated in a constant temperature water bath . After the reaction, the substrates were separated from the solution and washed with ultra-pure water to obtain the substrates with ZnO nanostructures.As shown in A field emission scanning electron microscope was used to examine the surface morphology of the samples as they were developed . Chemical component was characterized by energy dispersive spectrometry . Data-physics was used to characterize surface wettability, including water contact angle (WCA), oil contact angle (OCA), underoil–water contact angle (UWCA), and underwater–oil contact angle (UOCA) . The liquid droplets utilized for WCA, UWCA, CA and UOCA analyses had a volume of 4 μL. The amount of water remained in the collected filtrates was determined by Karl Fischer Titrator . Total organic carbon (TOC) equipment was used to measure the oil content of the filtrates  Global Laboratory Consumables Co. Ltd., Shanghai, China). The Zetasizer Nano ZS  was used to investigate the dispersion of water droplets in oil and oil droplets in water. The microscopy pictures of W/O and O/W emulsions were acquired by using an inverted microscope .C0 represented the dispersed phase content in the initial W/O and O/W emulsions (ppm); C1 represented the water/oil content in the collected filtrate (ppm); and η represented the separation efficiency. Meanwhile, the filtration flux (L) was calculated by:m was the mass of collected filtrate measured with a balance scale; ρ was the density of water/oil (ρ = 103 kg/m3); r was the radius of a glass tube (the radius of the glass tube in this study was 8 mm); t was the filtration duration, with the filtration flux computed by measuring the quantity of filtrate 1 min per time.Here, 99.6 g of infused oils  were combined with 0.4 g of water and ultrasonically agitated for 1 min to create water-in-oil(W/O) emulsions. To prepare oil-in-water (O/W) emulsions, 0.4 g of oil  was combined with 99.6 g of ultra-pure water and ultrasonically agitated for 10 min. The experiment of separation was performed by using a home-made filter device that sandwiched the created membrane between two glass tubes. Before assembling the separation device, the prepared filter membranes were placed in ultrapure water or oil solutions for 30 s, dependent on the types of emulsions. The pre-wetted filter membranes were used in the separation experiments. A glass cylinder tube containing metal fiber felt as a filter was placed at the position shown in − ions. The generated OH− ions form insoluble ZnO precipitated at supersaturated Zn(OH)42− ions or Zn2+ ions. As a polar crystal, ZnO (0001) exhibited a positive charge on the crystalline surface and attracted OH− ions and Zn(OH)42− ions in the solution. As a result, ZnO had the highest growth rate along the C-axis compared to other crystal planes and eventually forms ZnO nanostructures [The seed layer was coated on a metal fiber felt substrate using the sol-gel method. We fabricated ZnO-coated metal fiber felts by immersing the metal fiber felts with the seed layer into the precursor solution for the liquid phase growth process . At the ructures ,38,39,40SEM images indicated that the raw stainless-steel fiber felt was composed of metal fibers with an intricate arrangement and the surface of the metal fibers was smooth a,b. The The liquid-phase ZnO growth had two stages: dissolution and crystallization. The growth solution concentration was one of the critical parameters for ZnO’s nucleation and growth. The effect of growth solution concentration on the formation of ZnO coating on filter membrane was shown in The contact angle measurements were accustomed to analyzing the surface wettability of as-prepared filter membranes, as illustrated in In contrast to a stratified oil–water mixture, an oil-in-water emulsion containing emulsified oil droplets with various sizes was difficult to be separated. To prevent emulsified droplet penetration and achieve separation of O/W emulsions, the micropore size was one of the important characteristics of separation membranes. Here, the capacity of ZnO coated filter membrane to separate in the O/W emulsion was demonstrated. To analyze the separation efficacy of an as-prepared filter membrane, emulsions of diesel/water, N-decane/water, dodecane/water, and N-tetradecane/water were invented. −2·h−1 was obtained for diesel-in-water emulsion. These findings suggested that the ZnO-coated filter membrane could separate a variety of O/W emulsions. Though the separation efficiency obtained in this study was similar with previous studies using inorganic coating materials (Nano-ZnO and Nano-TiO2) to separate oil-in-water emulsions, we obtained a higher separation flux performance than these studies [Furthermore, other emulsions such as diesel/water, N-decane/water, and N-tetradecane/water were successfully separated a,b. As e studies ,42,43. IThe ZnO-coated filter membrane could separate O/W emulsion and O/W emulsion. The water content in the filtrate, separation flux, and corresponding separation efficiency were used to analyze the separation performance of the as-prepared filter membrane. As demonstrated in −2·h−1, respectively  could separate diverse water-in-oil and oil-in-water emulsions with a separation efficiency higher than 98%. Based on the oil–water separation mechanism, we could predict that the blocking of filter materials was caused by the collection of emulsified impurities. Thus, the effect of filtering quantity on the separation efficacy was investigated to analyze the influence of impurities. The investigation results showed that the filtration flux decreased with the collection of emulsified impurities. However, the filtration flux could restore after cleaning and drying, suggesting the recyclable nature of our strategy. The nanostructured filter material is a promising candidate for separating both water-in-oil and oil-in-water emulsion in industry. The proposed filter materials in this study may be repetitively used without any treatments, secondary pollution, and low energy consumption. Therefore, our methods may have wide application in the future.


Hemoperfusion is a method of blood filtering to remove toxins and inflammatory factors. Cytokine storms and high levels of inflammatory factors play a role in the pathogenesis of coronavirus disease 2019 (COVID-19). The present study aims to evaluate the effect of hemoperfusion on the clinical and laboratory findings and outcomes of patients with severe COVID-19. Forty-eight patients with severe COVID-19 and a positive PCR test, who were admitted to the intensive care unit (ICU), participated in the study. All patients were treated by routine treatment protocol for COVID-19. Hemoperfusion was performed for 24 patients in addition to treatment with conventional antiviral therapies. The other 24 patients made up the control group. Demographic data, laboratory findings and patient outcomes before and after treatment were retrospectively collected and analysed. There was no significnt difference in mortality or length of hospital stays between the control group and the hemoperfusion group. The breathing rate  and heart beat rate  of patients decreased after hemoperfusion. The hemoperfusion resulted in a significant increase in the SpO2 levels and a significant decrease in the CRP of patients compared to the conventional treatment . Hemoperfusion can improve respiratory distress. It can reduce the CRP in patients with severe COVID-19 but has no effect on mortality. Coronavirus disease 2019 (COVID-19) is an infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This acute viral infection is often associated with respiratory failure, pneumonia, acute respiratory distress syndrome (ARDS) and sepsis [Although most patients with COVID-19 have a mild form of the disease (81%), it is accompanied by severe and life-threatening manifestations with a mortality rate of 49%. Studies have shown that the levels of various cytokines such as IL-2R, IL-6, IL-10, IL-2, IL-7, GCSF and TNF-α are significantly higher in patients with severe COVID-19 compared with patients who have a mild disease ,4. MortaHemoperfusion is a method of filtering blood. The target molecules are removed from the blood when it passes through a column that contains adsorbent particles, which are either activated charcoal or resin. In this method, toxins or some inflammatory mediators are removed from the blood using special cartridges. Hemoperfusion can decrease IL-6 and IL-8 levels in patients with sepsis and can decrease the mortality rate .Inflammatory cytokines in patients with severe COVID-19 are high; therefore, hemoperfusion may improve the patients’ condition. Several studies have evaluated the therapeutic effect of hemoperfusion on COVID-19 patients and have obtained varying findings . FurtherThis retrospective study was carried out on patients with severe COVID-19 admitted to Shahid Beheshti Hospital in Kashan between February 2020 to March 2020. The Ethics Committee of Kashan University of Medical Sciences approved the study (IR.KAUMS.MEDNT.REC.1399.117). Patients admitted to ICU with severe COVID-19, including a positive PCR test result and a positive chest CT scan, participated in the study. All patients were treated by routine treatment protocol for COVID19. The patients were divided into two groups. The first group underwent hemoperfusion in the inflammatory phase of the disease in addition to the current treatment, while the second group only received conventional treatment. The patients underwent hemoperfusion three times with HA330 and HA280 filters for four hours. Inclusion criteria were: (1) having at least two clinical criteria, including respiratory rate more than 30 times per minute, oxygen saturation (SPO2) less than 85%, or intermittent fever.(2) having at least three laboratory criteria, including PaO2<60 mmHg, PaO2/FiO2<200, CRP>60 mg/dL, ferritin> 2000, an elevated D-dimer, fibrinogen <150 mg/dL and bicytopenia . All patients with a platelet count below 20,000 were excluded from the study.Laboratory tests were performed one day before and 72 hours after hemoperfusion. Demographic data, laboratory findings and outcomes of patients before and after treatment were retrospectively collected. To evaluate the effectiveness of hemoperfusion, PaO2, and vital signs before and after hemoperfusion (only in hemoperfusion group), as well as SpO2, ESR, CRP, fibrinogen, ferritin and lymphocytes of patients in both groups were extracted and compared.U test. Paired t-test and Wilcoxon signed-rank test were also used to evaluate the findings before and after the intervention in the hemoperfusion group. Normal distribution was checked using the Shapiro–Wilk test. A p-value of less than 0.05 was considered significant.Data analysed by SPSS version 22. Demographic characteristics and outcomes of patients between two groups were compared by Chi-square test, student’s t-test and Mann–Whitney Forty-eight patients with severe COVID-19 participated in this study. Twenty-four patients were placed in the hemoperfusion group, and the same number of patients were placed in the control group. The control group ranged in age from 33 to 97 years. The hemoperfusion group had an age range of 27 to 85 years. Demographic characteristics and outcomes of patients are summarized in Comparison of vital signs and PaO2 before and after hemoperfusion  showed tClinical and laboratory findings before and after treatment of patients with severe COVID-19 are summarized in Results of the present study showed a significant increase in the level of SpO2 and a significant decrease in the CRP level among the hemoperfusion group compared to the control group. In addition, a significant decrease in respiration rate, heart rate and an increase in the level of PaO2 was observed after hemoperfusion.Several studies have evaluated the therapeutic effect of hemoperfusion on COVID-19 patients. In the case of COVID-19 reported by Dastan et al., the patient underwent hemoperfusion three times with an HA380 cartridge. Subsequently, the level of SpO2 increased, and the patient’s clinical condition improved. The levels of IL-1, IL-6, IL-8 and TNF-α decreased . The finAlthough it is expected that hemoperfusion decreases inflammatory cytokines, it could not reduce the mortality rate and level of IL-6 in patients with septic shock, as shown in the study by Schädler et al. . In the Several studies have shown that coronaviruses can induce an excessive and prolonged cytokine/chemokine response (cytokine storm), which results in acute lung injury (ALI), acute respiratory distress syndrome (ARDS), high morbidity and mortality due to immunopathology. Previous studies have found an association between an increase in levels of IL-2, IL-6, IL-7, IL-10 and TNF-α and the severity or mortality of COVID-19. This evidence suggests the role of cytokine release syndrome (CRS) in disease severity and the recovery of patients. Therefore, it seems that the CRS can play an essential role in the pathogenesis of COVID-19. Hence, hemoperfusion can benefit patients with COVID-19 by removing these cytokines from the body and inhibiting CRS. It prevents lung tissue damage by preventing apoptosis of lung cells and increases the SpO2 level. Following patients’ improvement and inflammation reduction, ferritin and CRP levels are also reduced.There are several drugs that inhibit some of the cytokines, such as Tocilizumab (IL-6 receptor inhibitor) and anakinra (IL-1β inhibitor). However, the use of other alternative methods such as hemoperfusion is useful when patients are in critical condition. Anti-cytokine drugs act specifically on one cytokine, but hemoperfusion can decrease different cytokines simultaneously.The present study had several limitations. In this retrospective study, the level of inflammatory cytokines such as IL-6 was not measured at the time of intervention. Other factors influencing mortality, such as comorbidities, have not been considered.Hemoperfusion could improve respiratory distress and reduced the CRP in patients with severe COVID-19 but has no effect on mortality. It is recommended that further studies are performed with larger sample sizes.Alireza Soleimani: Conceptualization, Methodology. Sara Hasibi-Taheri: Data curation, Writing- Original draft preparation. Amir Hossein Loghman: Visualization, Investigation. Seyed-Masoud Moeini Taba: Supervision. Mohammad Shayestehpour: Writing- Reviewing and Editing.The authors declare that there is no conflict of interest.Kashan University of Medical Sciences (grant: 99126).This study was funded and supported by This study was approved by the Ethics Committee of Kashan University of Medical Sciences (IR.KAUMS.MEDNT.REC.1399.117).


Background: Previous studies neglected death as a critical competing risk while estimating the cancer risk for digoxin users. Therefore, the current study aims to assess the effectiveness of digoxin on cancer prevention by competing risk analysis.Methods: We performed a population-based retrospective cohort study using the Taiwan National Health Insurance Research database between 1998 and 2010. After one-to-one propensity score-matching from 36,160 patients with defined criteria, we enrolled 758 patients both in digoxin and β-blocker group for further analysis.Results: The results showed that the digoxin group had higher all-cause mortality than the β-blocker group in the 4- year (10.4 vs. 4.9%) and 8 years (13.6 vs. 7.0%) follow-up. The subdistribution HR of cancer incidence in the digoxin group compared to the β-blocker group was 1.99  and 1.46 (95% CI: 1.01–2.15) in the 4 years and 8 years follow-up, respectively.Conclusions: The result of our study showed the usage of digoxin has no benefit in cancer prevention compared with β-blocker. The possibility of β-blocker as a new drug candidate for cancer prevention needs further clinical evaluation. The current study also emphasized the necessity of competing risk analysis applying to similar clinical researches. Food and Drug Administration  randomly selected from NHI beneficiaries in Taiwan. Because the prescriptions of digoxin have been declining steadily over the last 15 years, which may have caused selection bias , we usedWe searched the records in NHIRD from 1998–2010 to identify patients aged ≥20 years with newly diagnosed HF, AF or AFL in our study. HF, AF, or AFL patients were identified by the International Classification of Diseases Revision, Ninth Revision, Clinical Modification (ICD-9-CM) codes, where 428 is for HF, 427.31 code is for AF, and 427.32 code is for AFL. To enhance diagnostic validity, patients with at least three consistent diagnoses of HF, AF, or AFL from outpatient medical records were enrolled in our study . We targTo enhance the validity of the current study, we made some exclusion criteria as follows : FirstlyTo reduce demographic differences between the digoxin group and the β-blocker group, we performed propensity-score matching by age, sex, medical comorbidities, medication history and the cDDD of target medications . The medThe primary outcome of the current study was cancer incidence (ICD-9-CM: 140–208), which was identified from the inpatient medical records. As we mentioned above, death was a competing risk event because its occurrence cannot be treated as independent censoring when analyzing the time to cancer occurrence. Therefore, the in-hospital mortality was identified as a competing risk event from the inpatient medical records using NHIRD. The current study was approved by the Institutional Review Board of Antai Medical Care Cooperation Antai-Tian-Sheng Memorial Hospital in 2018.Descriptive statistics were used to assess patients’ demographics. The standardized mean differences were calculated for the matched cohort to assess balance in measured baseline covariates . Some reAs shown in After 1:1 propensity-score matching by criteria, there were 758 patients in the digoxin group and 758 patients in the β-blocker group. The cancer-related covariates were well-balanced after matching . The meaAfter 1:1 propensity-score matching by covariates, the all-cause mortality was statistically higher for the digoxin group (10.4 vs. 4.9%) in the 4 years follow-up and 8 years follow-up (13.6 vs. 7.0%) . Competi+/K+-ATPase can function as a signal transducer, the opportunity of repurposing digoxin as well as the development of other cardiac glycosides as anti-cancer drugs have increased interest in previous studies  . HoweverAlthough cardiac glycosides decrease expression of prostate-specific antigen by down-regulation of prostate-derived Ets factor , digoxinAlthough the study generated significant findings, our results should be interpreted with caution. Firstly, the results are limited to patients with HF, AF or AFL. The effect of digoxin on healthy participants could not be obtained in the NHIRD study. Secondly, although we used multiple strategies to minimize confounding by propensity-score matching, the current observational study might have residual confounding factors and cannot prove causality. Third, the medical adherence and some time-varying changes might influence the result. Therefore, the result was less evident in 8 years than 4 years follow up. Finally, the incidence of specific cancer types was not evaluated because the cancer case numbers were too low for advance analysis.in vitro, for now, it is virtually impossible to perform a clinical trial to evaluate the association of digoxin and cancer incidence for the healthy population because of the safety issues of digoxin. We suggest that researchers can screen natural cardiac glycosides with the same or better anti-cancer effects but lower cardio-inotropic effects compared with digoxin.Although digoxin proves its anti-cancer effects in vitro linked to cancer growth inhibition through impaired production of vascular endothelial growth factor (VEGF)  , decreasr (VEGF) , and enhr (VEGF) . One obsr (VEGF) , while ar (VEGF) . HoweverOur study is the first to include the death event as the competing risk while evaluating the association of digoxin and cancer incidence. Our results suggest traditional Cox regression model underestimated the risk. After considering the risk of death, digoxin users had a higher risk of cancer incidence compared to β-blockers. The current study demonstrated the necessity of competing risk analysis applying to similar clinical researches.For clinical use for cancer prevention, the assessment of digoxin or other natural derived cardiac glycosides should consider their safety issue first. Further studies are required to evaluate their effectiveness.